This research is aimed at further understanding of post-translational events in the in vivo formation of hemoglobin tetrameres. There are two important events: proper assembly of nascent globin chains or heme- containing subunits; and insertion of heme into the polypeptide chain. The investigator believes these events are important determinants of human hemoglobin phenotype expression. The proposed research consists of 5 specific aims: 1. Preparation and structural characterization of heme and globin subunit assembly intermediates, and exon-encoded domain peptide regions. These isolates will enable the investigator to evaluate all plausible pathways of human hemoglobin assembly. 2. Kinetic evaluation of hemoglobin formation from normal and variant heme subunits. The investigators will study, using kinetic techniques, the assembly of hemoglobin subunits isolated from both normal and mutant proteins. This is expected to lead to a better understanding of the critical regions of the alpha and beta polypeptides that determine combination parameters. 3. Kinetic studies of the role of globin intermediates in hemoglobin assembly. Using fluorescence stopped-flow, the investigators will study the association of globin (alpha-o/beta-o) and heme-containing globin (alpha-h/beta-h) into semihemoglobins (alpha-h/beta-o, alpha-o/beta-h), which are known intermediates in the assembly process. 4. Kinetic investigation of heme insertion into globin and semihemoglobin proteins. Previous studies of heme-globin association rates will be extended using stopped-flow techniques in an effort to discover to what extent this reaction is a """"""""molecular governess"""""""" of alpha beta coupling. 5. Interaction of the band 3:cytoplasmic domain protein with assembly intermediates. The interaction of intact hemoglobin tetrameres and isolated alpha and beta subunits with the cytoplasmic domain of band 3 (CDB3) membrane component will be studied to determine whether tetramere stabilization impacts on hemoglobin subunit assembly.
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